This invention relates to a novel polymer thin film adapted for use in the fields of laboratory testing, medicine, and the like; its production method; and a bio chip utilizing such polymer thin film which is adapted for use in assaying gene expression, gene mutation, gene polymorphism, and the like.
Some devices such as a catheter employed in the field of medicine are used by insertion into a body. Introduction of a medical device which is a foreign matter to the body is associated with the problem of compatibility with the biological systems in the body such as immune and defense mechanisms of the body.
For example, thrombosis is one of the most serious problems associated with the development and use of medical devices such as blood collecting and treating systems. When the blood becomes in contact with the surface of a foreign matter, the liquid content and the cells of the blood undergo some change. To improve the biocompatibility of such devices, immobilization of an anticoagulant or immobilization of a biologically active antithrombogenic agent on the polymer surface is required.
Expression of genes in cells and tissues has been assayed by Northern blotting (or dot blotting) wherein RNA from various cells or tissues is immobilized on the membrane, and the RNA is hybridized by using a probe specific to the analyte gene; RT-PCR using a primer specific to the analyte gene; or the like.
There is also a demand for an assay wherein a large number of genes are assayed at once reflecting the progress in gene researches and associated increase in the number of analyte genes as well as progress of the Genome Project and application of its outcome in the field of medicine.
In view of such demands, various techniques have been developed that utilize microarray, DNA chip, and the like. Such techniques share the common feature that several thousands DNA fragments of different types are immobilized on a glass substrate (which is referred to as a DNA chip or a bio chip), and the target DNA fragment is detected at a high sensitivity by means of a reaction between the immobilized DNA fragment and the very minute amount of labeled target DNA fragment.
Such procedures have enabled to assay a large number of genes of human and other mammals or even the entire genes of a microorganism having several thousand genes on several bio chips. Also enabled are assays of the amount gene expression for the entire genes by using labeled RNAs. Assay of mutation such as gene deletion has also been enabled by labeling the genomic DNA.
When a bio chip is produced by a procedure other than the “on chip” synthesis (i.e. the procedure wherein the DNA fragments to be immobilized on the surface of the substrate are directly synthesized on the surface of the substrate), the DNA fragments that had been produced beforehand are spotted on the surface of the substrate, and immobilized by utilizing electrostatic interactions or covalent bonding.
FIG. 2 is a view explaining the principle of this procedure. FIG. 2 STEP (A) shows a microplate 22 with probe DNAs 21 of different types placed in the microplate. In the meanwhile, a glass plate as shown in FIG. 2 STEP (B) is prepared for uses as a plate 23, and as shown FIG. 2 STEP (C), the surface of the plate 23 is coated with a binder 24 such as poly-l-Lysine which binds the DNA to the glass. Next, the probe DNA 21 in the microplate 22 is attached to a pin, and the DNA 21 attached on the pin is brought in contact with the glass plate 23 that had been coated with the binder (poly-l-Lysine) 24 of the DNA and the glass for spotting of the DNA on the coated glass. This procedure is repeated until the spotting of all probe DNA in the microplate 22 has been completed, and a bio chip shown in FIG. 2 STEP (D) is thereby produced. As described above, bio chips have been produced by preliminarily coating the entire surface of a plate with a binder of the DNA and the glass, and thereafter spotting the DNA on the plate coated with the binder.
The hybridization of the bio chip is accomplished by placing the bio chip wherein the probe DNAs have been spotted on the glass plate by means of the binder with the sample DNA that had been labeled with a fluorescent substance in a hybridization solution to thereby promote the hybridization. The hybridization solution is a mixed solution comprising formaldehyde, SSC (NaCl, trisodiumcitrate), SDS (sodium dodecyl sulfate), EDTA (ethylenediamidetetraacetic acid), distilled water, and the like, and mixing ratio may vary depending on the character of the DNA used.
In this step, the sample DNA and the probe DNA will bind to each other on the bio chip by forming a double helix structure if these DNAs have complementary strands. On the other hands, the DNAs will not bind to each other if the DNAs are not complimentary to each other, and the sample DNA that had been labeled with a fluorescent substance either remains in the hybridization solution or becomes bound to the binder coated on the glass plate to remain as a garbage.
When the glass plate is washed in a water tank or the like to thereby remove the sample DNA that had been labeled with a fluorescent substance remaining on the glass plate, the sample DNA that failed to bind to the probe DNA is washed away. The hybridization is then detected by exciting the fluorescent substance that had been used for labeling the sample DNA and that became bonded to the probe DNA by the light energy emitted from the predetermined light source, and scanning the light emitted by the excitation of the fluorescent substance using a photosensor such as CCD.
However, the binder of the DNA and the glass such as poly-l-Lysine is insufficient in the binding strength with the DNA, and the probe DNA often became detached from the substrate together with the hybridized sample. The loss of the probe DNA and the sample DNA due to such insufficient binding often reached as high as 70% mass ratio, and it has been the state of art that the expensive probe DNA and the precious sample DNA are being wasted.
In order to obviate such problem, various materials have been examined for use as a binder. In spite of such attempts, no material has so far been found effective.